Search

The proliferation of antibiotic usage has precipitated the emergence of drug-resistant variants of bacteria, thereby augmenting their capacity to withstand pharmaceutical interventions. Among these variants, Cronobacter sakazakii (C. sakazakii), prevalent in powdered infant formula (PIF), poses a grave threat to the well-being of infants. Presently, global contamination by C. sakazakii is being observed. Consequently, research endeavors have been initiated to explore the strain's drug resistance capabilities, alterations in virulence levels, and resistance mechanisms. The primary objective of this study is to investigate the resistance mechanisms and virulence levels of C. sakazakii induced by five distinct antibiotics, while concurrently conducting transcriptomic analyses. Compared to the susceptible strains prior to induction, the drug-resistant strains exhibited differential gene expression, resulting in modifications in the activity of relevant enzymes and biofilm secretion. Transcriptomic studies have shown that the expression of glutathione S-transferase and other genes were significantly upregulated after induction, leading to a notable enhancement in biofilm formation ability, alongside the existence of antibiotic resistance mechanisms associated with efflux pumps, cationic antimicrobial peptides, and biofilm formation pathways. These alterations significantly influence the strain's resistance profile. [ABSTRACT FROM AUTHOR]

The proliferation of antibiotic usage has precipitated the emergence of drug-resistant variants of bacteria, thereby augmenting their capacity to withstand pharmaceutical interventions. Among these variants, Cronobacter sakazakii (C. sakazakii), prevalent in powdered infant formula (PIF), poses a grave threat to the well-being of infants. Presently, global contamination by C. sakazakii is being observed. Consequently, research endeavors have been initiated to explore the strain’s drug resistance capabilities, alterations in virulence levels, and resistance mechanisms. The primary objective of this study is to investigate the resistance mechanisms and virulence levels of C. sakazakii induced by five distinct antibiotics, while concurrently conducting transcriptomic analyses. Compared to the susceptible strains prior to induction, the drug-resistant strains exhibited differential gene expression, resulting in modifications in the activity of relevant enzymes and biofilm secretion. Transcriptomic studies have shown that the expression of glutathione S-transferase and other genes were significantly upregulated after induction, leading to a notable enhancement in biofilm formation ability, alongside the existence of antibiotic resistance mechanisms associated with efflux pumps, cationic antimicrobial peptides, and biofilm formation pathways. These alterations significantly influence the strain’s resistance profile.

Cronobacter sakazakii can cause severe illnesses in infants, predominantly in preterm newborns, with consumption of contaminated powdered infant formula (PIF) being the major vehicle of infection. Using a dynamic human gastrointestinal simulator called the SHIME, this study examined the effects of gastric acidity and gastric digestion time of newborns on the survival and expression of stress genes of C. sakazakii. Individual strains, inoculated at 7 log CFU/mL into reconstituted PIF, were exposed to gastric pH values of 4.00, 5.00 and 6.00 for 4 h with gradual acidification. The survival results showed that C. sakazakii grew in the stomach portion of the SHIME during a 4-h exposure to pH 4.00, 5.00 and 6.00 by 0.96–1.05, 1.02–1.28 and 1.11–1.73 log CFU/mL, respectively. The expression of two stress genes, rpoS and grxB, throughout gastric digestion was evaluated using reverse transcription qPCR. The upregulation of rpoS and grxB during the 4-h exposure to simulated gastric fluid at pH 4.00 showed that C. sakazakii strains may be experiencing the most stress in the pH 4.00 treatment. The gene expression results also suggest that C. sakazakii strains appeared to develop an acid adaptation response during the 4-h exposure that may facilitate their survival. Altogether, this study highlights that a combination of low gastric acidity, long digestion time in the presence of reconstituted PIF, created a favorable environment for the adaptation and survival of C. sakazakii in the simulation of a newborn’s stomach. This study gives directions for future research to further advance our understanding of the behavior of C. sakazakii in the GI tract of newborns.

The occurrence of Cronobacter spp. was investigated in table eggs and egg-based desserts obtained from retail stores. Cronobacter was isolated from 57 out of 180 (31.7%) (95% Confidence Interval [CI]: 26--43%) examined samples of eggs and egg-based desserts. The prevalence of Cronobacter spp. was significantly higher in farm eggs (67%) (P < 0.05) than in Balady eggs (23%), cream cake (27%) and small-scale ice cream (27%). Cronobacter (C.) sakazakii and C. muytjensii were the predominant isolates obtained in the present study. All C. sakazakii isolates were molecularly confirmed with higher incidence in small-scale ice cream (62.5%). [ABSTRACT FROM AUTHOR]

ABSTRACT: Cronobacter sakazakii is a harmful foodborne pathogen, and its contaminated food will pose a huge threat to human health. Prevention of C. sakazakii contamination of food is valuable for food safety as well as for human health. In this study, silver nanoparticles (AgNP) were successfully immobilized on the surface of cellulose acetate (CA) and polymethylmethacrylate (PMMA) composite to obtain AgNP/PMMA/CA film. Through the inhibition zone and growth curve experiments, we found that AgNP/PMMA/CA films has excellent antibacterial activity on C. sakazakii. The AgNP/PMMA/CA film can prolong the lag phase of the growth curve of C. sakazakii from 2 to 8 h. The antibacterial films were found to reduce the survival of C. sakazakii in Luria-Bertani and infant formula by combining it with a mild heat treatment (45°C, 50°C, and 55°C). The AgNP/PMMA/CA film combined with 55°C water bath can completely inactivate C. sakazakii in infant formula within 120 min. Finally, the potential mechanism by which AgNP/PMMA/CA films reduce the heat tolerance of C. sakazakii was investigated by quantitative real-time PCR. The results showed that AgNP/PMMA/CA films could reduce the expression of environmental tolerance-related genes in C. sakazakii. The current research shows that AgNP/PMMA/CA film has strong antibacterial activity, and the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens. The AgNP/PMMA/CA film can be used as a potential packaging material or antibacterial surface coating.

The present work developed an electrochemical genosensor for the detection of virulence outer membrane protein A (ompA, tDNA) gene of Cronobacter sakazakii (C. sakazakii) by exploiting the excellent glucose-oxidase-mimicking activity of copper Metal-organic frameworks (Cu-MOF) doped with gold nanoparticle (AuNPs). The signal nanotags of signal probes (sDNA) that biofunctionalized AuNPs@Cu-MOF (sDNA-AuNPs@Cu-MOF) were designed using an Au-S bond. The biosensor was prepared by immobilization capture probes (cDNA) onto an electrodeposited AuNPs-modified glassy carbon electrode (GCE). AuNPs@Cu-MOF was introduced onto the surface of the GCE via a hybridization reaction between cDNA and tDNA, as well as tDNA and sDNA. Due to the enhanced oxidase-mimicking activity of AuNPs@Cu-MOF to glucose, the biosensor gave a linear range of 1.0 × 10−15 to 1.0 × 10−9 mol L−1 to tDNA with a detection limit (LOD) of 0.42 fmol L−1 under optimized conditions using differential pulse voltammetry measurement (DPV). It can be applied in the direct detection of ompA gene segments in total DNA extracts from C. sakazakii with a broad linear range of 5.4−5.4 × 105 CFU mL−1 and a LOD of 0.35 CFU mL−1. The biosensor showed good selectivity, fabricating reproducibility and storage stability, and can be used for the detection of ompA gene segments in real samples with recovery between 87.5% and 107.3%. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii is a harmful foodborne pathogen, and its contaminated food will pose a huge threat to human health. Prevention of C. sakazakii contamination of food is valuable for food safety as well as for human health. In this study, silver nanoparticles (AgNP) were successfully immobilized on the surface of cellulose acetate (CA) and polymethylmethacrylate (PMMA) composite to obtain AgNP/PMMA/CA film. Through the inhibition zone and growth curve experiments, we found that AgNP/PMMA/CA films has excellent antibacterial activity on C. sakazakii. The AgNP/PMMA/CA film can prolong the lag phase of the growth curve of C. sakazakii from 2 to 8 h. The antibacterial films were found to reduce the survival of C. sakazakii in Luria-Bertani and infant formula by combining it with a mild heat treatment (45°C, 50°C, and 55°C). The AgNP/PMMA/CA film combined with 55°C water bath can completely inactivate C. sakazakii in infant formula within 120 min. Finally, the potential mechanism by which AgNP/PMMA/CA films reduce the heat tolerance of C. sakazakii was investigated by quantitative real-time PCR. The results showed that AgNP/PMMA/CA films could reduce the expression of environmental tolerance-related genes in C. sakazakii. The current research shows that AgNP/PMMA/CA film has strong antibacterial activity, and the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens. The AgNP/PMMA/CA film can be used as a potential packaging material or antibacterial surface coating. [ABSTRACT FROM AUTHOR]

Aims: The aim was to characterize a collection of Cronobacter sakazakii isolates collected from various origins in Jordan. Methods and Results: The isolates were characterized using 16S rRNA sequencing, DNA microarray, multi‐locus sequence typing (MLST), O‐serotyping, virulence gene identification and antibiotic susceptibility testing. The identities and phylogenetic relatedness revealed that C. sakazakii sequence type 4 (ST4) and Csak O:1 serotype were the most prevalent STs and serovars amongst these C. sakazakii strains. PCR screening of putative virulence genes showed that the siderophore‐interacting protein gene (sip) and iron acquisition gene clusters (eitCBAD and iucABCD/iutA) were the most detected genes with noticeable variability in the type 6 secretion system (T6SS) and filamentous hemagglutinin/adhesion (FHA) gene loci. The antibiotic resistance profiles revealed that the majority of the isolates were susceptible to all antibiotics used despite harbouring a class C β‐lactamase resistance gene. Conclusions: The results described in this report provide additional insights about the considerable genotypic and phenotypic heterogeneity within C. sakazakii. Significance and Impact of the Study: The information reported in this study might be of great value in understanding the origins of C. sakazakii isolates, in addition to their diversity and variability, which might be helpful in preventing future outbreaks of this pathogen. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii is associated with the ingestion of contaminated reconstituted powdered infant formula (PIF), resulting in necrotizing enterocolitis, sepsis and meningitis in neonatal infants. Potential virulence determinants include the variable capsular polysaccharides; K-antigen and colanic acid (CA). Strains encoding for the capsule variant K2:CA2 have been strongly associated with neonatal meningitis cases. This study aimed to develop and apply a multiplex PCR assay to determine C. sakazakii K-antigen and colanic acid types. Twenty-six strains of C. sakazakii which had previously been isolated from food and environmental sources were used. These cover 18 multilocus sequence types and four serotypes. Based on our research findings, we have identified two K-antigen types present. Specifically, the K1-antigen was observed in sequence types ST1, ST8, ST20, ST23, ST64, ST198, ST263, ST264 and ST406, while the K2-antigen was present in ST4, ST9, ST12, ST13, ST136, ST233, ST245 and ST405. Additionally, we detected colanic acid (CA) type 1 in sequence types ST1, ST8, ST9, ST20, ST245 and ST405, and colanic acid (CA) type 2 in ST4, ST12, ST13, ST23, and ST64. We compared the predicted K-antigen and colanic acid types with the entire genome sequences of the strains. The comparison showed complete agreement between the PCR amplification results and the genomic analysis of the K-antigen and colanic acid-encoding regions. This assay is a useful tool for rapid identification of C. sakazakii , K-antigen and colanic acid types, in routine diagnoses and foodborne investigations. In addition, it will contribute to our knowledge of virulence factors associated with life-threatening neonatal meningitis.

This study aims to detect the prevalence and antimicrobial resistance of Listeria monocytogenes and Cronobacter sakazakii in three dairy households and dried milk from different suppliers, and evaluate the antimicrobial effect of rose water, rose, and orange essential oils. In total, 360 samples were collected from cattle, the environment, and dried milk (n = 30). Antimicrobial activity was evaluated with twofold microtube dilution and the time-kill method. L. monocytogenes was identified in all households (13.3%) with a prevalence in the range of 5.8–17.5%, while C. sakazakii was identified in one household (5.3%). The former and latter pathogens were highly isolated from the feces at 20% and 2.5% and bedding at 12.5% and 1.6%, respectively. L. monocytogenes was isolated only from milk at 7.5%, but C. sakazakii was not detected in either milk or dried milk. L. monocytogenes strains were screened for virulence genes (iap, hylA, and actA). All strains were positive for the iap gene, while for hlyA and actA, the percentages were (35.4% 16.6%, respectively). L. monocytogenes strains showed high resistance against sulfamethoxazole–trimethoprim (100%), followed by gentamicin, penicillin, and imipenem (95.8%, 95.8%, and 91.6%, respectively). All C. sakazakii strains were susceptible to all tested antibiotics. The bactericidal activity of orange oil was the strongest, appeared after 1 h for both pathogens, followed by rose oil and then rose water. [ABSTRACT FROM AUTHOR]

The quorum-sensing receptor SdiA is vital for regulating the desiccation tolerance of C. sakazakii , yet the specific mechanism remains elusive. Herein, transcriptomics and phenotypic analysis were employed to explore the response of C. sakazakii wild type (WT) and sdiA knockout strain (Δ sdiA) under drying conditions. Following 20 days of drying in powdered infant formula (PIF), WT exhibited 4 log CFU/g higher survival rates compared to Δ sdiA. Transcriptome revealed similar expression patterns between csrA and sdiA , their interaction was confirmed both by protein-protein interaction analysis and yeast two-hybrid assays. Notably, genes associated with flagellar assembly and chemotaxis (flg , fli , che , mot regulon) showed significantly higher expression levels in WT than in Δ sdiA , indicating a reduced capacity for flagellar synthesis in Δ sdiA , which was consistent with cellular morphology observations. Similarly, genes involved in trehalose biosynthesis (ostAB, treYZS) and uptake (thuEFGK) exhibited similar expression patterns to sdiA , with higher levels of trehalose accumulation observed in WT under desiccation conditions compared to Δ sdiA. Furthermore, WT demonstrated enhanced protein and DNA synthesis capabilities under desiccation stress. Higher expression levels of genes related to oxidative phosphorylation were also noted in WT, ensuring efficient cellular ATP synthesis. This study offers valuable insights into how SdiA influences the desiccation tolerance of C. sakazakii , paving the way for targeted strategies to inhibit and control this bacterium. • CsrA interacts with SdiA, exhibiting similar expression patterns. • Flagella play a crucial role in the desiccation tolerance of the wild type strain. • Accumulation of osmoprotectants enhances desiccation resistance in the wild type. • Protein synthesis ensures DNA integrity and energy supply under dry conditions. [ABSTRACT FROM AUTHOR]

The quorum sensing receptor SdiA is vital for the environmental resistance of Cronobacter sakazakii , facilitating its survival in powdered infant formula (PIF). In this study, a three-dimensional model of the SdiA receptor was developed, and molecular docking was used to screen inhibitors targeting SdiA. The interaction mechanisms and the blocking effect of inhibitors on quorum sensing were also validated. Among hit compounds, phloretin significantly reduced the environmental resistance of the wild type (WT) at a sub-inhibitory concentration of 0.6 mg/mL (p < 0.05), lowering the survival rates from 4.61, 5.65, and 5.32 log to 2.76, 1.30, and 1.71 log under heat, osmotic, and desiccation stress, respectively. In simulated aqueous conditions, the stability of phloretin-SdiA complex was maintained throughout over 70 ns, with van der Waals forces and electrostatic interactions crucial for its structural stability. The interactions between phloretin and SdiA lead to a discernible increase in the UV absorption and a significant quenching of the endogenous fluorescence of the SdiA protein, with the α-helical content increasing from 54.3% to 65.2%. Additionally, inoculation of 4 log 10 CFU/mL of WT in dry powder materials of PIF, combined with phloretin at a concentration of 0.6 mg/mL and simulation of evaporation and drying processes, reduced WT below detection limit for enumeration. The outcomes of this study provide valuable scientific evidence and insights for developing innovative prevention and control strategies against C. sakazakii. [Display omitted] • Phloretin exhibited a strong inhibitory effect on SdiA-Mediated quorum sensing. • UV, fluorescent and CD spectroscopy confirmed the interaction of phloretin with SdiA. • Van der Waals forces contributed to structural stability of SdiA-phloretin complex. • Phloretin inactivated C. sakazakii in whey protein and oligofructose effectively. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii (C. sakazakii), a foodborne pathogen frequently present in contaminated powdered infant formula (PIF), poses a substantial threat to food safety. Herein, a novel fluorescent biosensor based on gold nanoclusters (AuNCs) that exhibit aggregation-induced emission (AIE) when triggered by nucleic acids was developed. This biosensor integrates the exponential amplification reaction (EXPAR) with hybridization chain reaction (HCR) signal amplification (EXHCR), enabling ultrasensitive detection of C. sakazakii. The aggregations of glutathione protected AuNCs assembled by G-rich sequences (HP1, and HP2) was affected by the EXHCR reaction initiated with C. sakazakii , forming G-quadruplex nanowires, leading to the dis-aggregation of AuNCs and a turn-off response of fluorescence, thereby realizing the detection of C. sakazakii. The changes are discernible on cellulose paper with the naked eyes. The biosensor demonstrates exceptional selectivity due to the integration of an aptamer and a dual signal amplification strategy. Under optimized conditions, it achieves a remarkable detection limit of 1.10×100 CFU/mL, and a broad linear range from 1.10×100 to 1.10×107 CFU/mL. Furthermore, the biosensor's capability to identify C. sakazakii in artificially contaminated milk powder with recoveries ranging from 97.8% to 107%, underscores its reliability and accuracy. Overall, this study offers a promising approach for nucleic acid and AIE-based detection, exhibiting considerable potential for developing highly efficient bacterial detectors. [Display omitted] • The EXHCR-AIE sensor exhibits excellent detection performance for C. sakazakii with a limit of 1.1×100 CFU/mL. • The study provided a specific connection idea for nucleic acid and the generation of AIE. • The C. sakazakii can be identified on cellulose paper by naked eyes. [ABSTRACT FROM AUTHOR]

The present work developed an electrochemical genosensor for the detection of virulence outer membrane protein A (ompA, tDNA) gene of Cronobacter sakazakii (C. sakazakii) by exploiting the excellent glucose-oxidase-mimicking activity of copper Metal-organic frameworks (Cu-MOF) doped with gold nanoparticle (AuNPs). The signal nanotags of signal probes (sDNA) that biofunctionalized AuNPs@Cu-MOF (sDNA-AuNPs@Cu-MOF) were designed using an Au-S bond. The biosensor was prepared by immobilization capture probes (cDNA) onto an electrodeposited AuNPs-modified glassy carbon electrode (GCE). AuNPs@Cu-MOF was introduced onto the surface of the GCE via a hybridization reaction between cDNA and tDNA, as well as tDNA and sDNA. Due to the enhanced oxidase-mimicking activity of AuNPs@Cu-MOF to glucose, the biosensor gave a linear range of 1.0 × 10−15 to 1.0 × 10−9 mol L−1 to tDNA with a detection limit (LOD) of 0.42 fmol L−1 under optimized conditions using differential pulse voltammetry measurement (DPV). It can be applied in the direct detection of ompA gene segments in total DNA extracts from C. sakazakii with a broad linear range of 5.4−5.4 × 105 CFU mL−1 and a LOD of 0.35 CFU mL−1. The biosensor showed good selectivity, fabricating reproducibility and storage stability, and can be used for the detection of ompA gene segments in real samples with recovery between 87.5% and 107.3%.

Silver nanoparticles (AgNPs) is an excellent antibacterial agent, which is widely used in medical, food, environmental and other fields, but AgNPs are easy to accumulate in aqueous solution, so their application in various fields is limited. Therefore, it is particularly important to propose a new application method or to prepare a new composite material. In this study, OA/PVA was obtained by cross-linking oxalic acid (OA) with polyvinyl alcohol (PVA). Then Ag/NCC was obtained by in situ reduction of AgNPs on nanocellulose crystals (NCC). Finally, Ag/NCC/OA/PVA composite antimicrobial films with good waterproofing effect were prepared by mixing Ag/NCC with OA/PVA. Subsequently, the films were characterized using SEM, UV–vis, FTIR and XRD, as well as physicochemical properties such as mechanical strength and hydrophilic properties were determined. The results indicated that the Ag/NCC/OA/PVA films possess good light transmittance, mechanical properties, water resistance, antibacterial activity, and biodegradability. The results of the mechanism study showed that Ag/NCC/OA/PVA films can destroy cell integrity, inhibit succinate dehydrogenase (SDH) activity, thereby reducing intracellular ATP levels. And induce a large number of reactive oxygen species (ROS) production, eventually leading to the death of C. sakazakii. In summary, Ag/NCC/OA/PVA film has good physical and chemical properties, antibacterial activity and biocompatibility, and has promising applications in food and medical antibacterial fields. • Ag/NCC/OA/PVA film has good degradability and biocompatibility. • Ag/NCC/OA/PVA film can inhibit the formation of bacterial biofilm. • Ag/NCC/OA/PVA film can reduce SDH activity and ATP synthesis in bacteria. • Ag/NCC/OA/PVA film can induce the increase of ROS content in bacteria. [ABSTRACT FROM AUTHOR]

This study aims to detect the prevalence and antimicrobial resistance of Listeria monocytogenes and Cronobacter sakazakii in three dairy households and dried milk from different suppliers, and evaluate the antimicrobial effect of rose water, rose, and orange essential oils. In total, 360 samples were collected from cattle, the environment, and dried milk (n = 30). Antimicrobial activity was evaluated with twofold microtube dilution and the time-kill method. L. monocytogenes was identified in all households (13.3%) with a prevalence in the range of 5.8–17.5%, while C. sakazakii was identified in one household (5.3%). The former and latter pathogens were highly isolated from the feces at 20% and 2.5% and bedding at 12.5% and 1.6%, respectively. L. monocytogenes was isolated only from milk at 7.5%, but C. sakazakii was not detected in either milk or dried milk. L. monocytogenes strains were screened for virulence genes (iap, hylA, and actA). All strains were positive for the iap gene, while for hlyA and actA, the percentages were (35.4% 16.6%, respectively). L. monocytogenes strains showed high resistance against sulfamethoxazole–trimethoprim (100%), followed by gentamicin, penicillin, and imipenem (95.8%, 95.8%, and 91.6%, respectively). All C. sakazakii strains were susceptible to all tested antibiotics. The bactericidal activity of orange oil was the strongest, appeared after 1 h for both pathogens, followed by rose oil and then rose water.

In the current study, we investigated the potential of Cronobacter sakazakii- ethylenediaminetetraacetic acid (EDTA) assisted phytoremediation potential of Zea mays L. to remediate lead (Pb)-contaminated soils. The C. sakazakii exhibited various stress tolerance mechanisms via plant growth promoting (PGP) traits, intrinsic extracellular enzyme production and antibiotic resistance. A greenhouse experiment was conducted to examine the dual effects of plant growth promoting endophytic bacteria (PGPEB)-chelator synergy in maize plants under different Pb contaminated soil regimes. C. sakazaii-EDTA (5 mM EDTA kg−1) complex significantly (p < 0.05) enhanced plant growth and biomass (48.91%); chlorophyll a, b and carotenoid contents (27.26%, 25.02% and 42.09%); relative water content (61.33%); proline content (63.60%); root and shoot Pb accumulation capacity (52.31% and 44.71%) in Pb contaminated soils. This may suggest the efficacy of current approach in enhancing plant tolerance capability toward Pb-uptake and phytoremediation capacity. Moreover, maize plants showed differential response to Pb availability in soil-1 (S1; Pb spiked soil, 500 mg kg−1) and soil-2 (S2; aged-contaminated soil) under various treatments. We describe the intriguing role of C. sakazakii-EDTA-maize system for Pb decontamination which can be used as a base line to explore the proposed combinatorial approach for long-term trails under field conditions for reclamation of Pb-contaminated soils. The PGPEB-EDTA mediated potential of Z. mays against Pb spiked and industrial contaminated soils is noticed. Increased tolerance of Z. mays against Pb in association with C. sakazakii, and EDTA is reported first time. Enhanced accumulation of metals by Z. mays is reported under combined treatment of C. sakazakii, and EDTA. Inoculation of plants with C. sakazakii, and EDTA has positive effects on growth and accumulation of Pb by Z. mays. [ABSTRACT FROM AUTHOR]

Fifteenth isolates of C. sakazakii were obtained from previous studies of the sample (infant formula, cerebrospinal fluid and blood). All isolates C. sakazakii identification based on microscopic, biochemical test and confirmed by 16SrRNA. We studied the movement of all isolates and study adhesion to polystyrene plate, adhesion and invasion to Esophageal adenocarcinoma (SKG-GT-4) for four isolates [Cerebrospinal fluid (CSF5), Bloods (B 1), Dialak (A1c), Novolac Allernova (C1)] and its cytotoxicity. Results showed that all isolates can move after 4 hours of incubation and increased after 8 hours, the isolates moved to different distances strong, medium, and weak. The results showed that the number of C. sakazakii colony adherent to polystyrene plate was more than 300 cfu for four isolates. while adhesion to (SKG-GT-4) the results showed the number of C. sakazakii colony adhesion to (SKG-GT-4) cell line in dilution (1 : 10 and 1 : 100) were more than 300 cfu to 4 isolated. Finally the colony numbers in the invasion SKG-GT-4, the results showed that the number was CSF5 (200cfu), A1C (183cfu), B1 (175cfu), C1 (290cfu). Cytotoxicity of C. sakazakii was less. In conclusion, C. sakazakii can move, adhesion to polystyrene, adhesion to, invasion to Esophageal adenocarcinoma and cytotoxic. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii can cause severe illnesses in infants, predominantly in preterm newborns, with consumption of contaminated powdered infant formula (PIF) being the major vehicle of infection. Using a dynamic human gastrointestinal simulator called the SHIME, this study examined the effects of gastric acidity and gastric digestion time of newborns on the survival and expression of stress genes of C. sakazakii. Individual strains, inoculated at 7 log CFU/mL into reconstituted PIF, were exposed to gastric pH values of 4.00, 5.00 and 6.00 for 4 h with gradual acidification. The survival results showed that C. sakazakii grew in the stomach portion of the SHIME during a 4-h exposure to pH 4.00, 5.00 and 6.00 by 0.96-1.05, 1.02-1.28 and 1.11-1.73 log CFU/mL, respectively. The expression of two stress genes, rpoS and grxB, throughout gastric digestion was evaluated using reverse transcription qPCR. The upregulation of rpoS and grxB during the 4-h exposure to simulated gastric fluid at pH 4.00 showed that C. sakazakii strains may be experiencing the most stress in the pH 4.00 treatment. The gene expression results also suggest that C. sakazakii strains appeared to develop an acid adaptation response during the 4-h exposure that may facilitate their survival. Altogether, this study highlights that a combination of low gastric acidity, long digestion time in the presence of reconstituted PIF, created a favorable environment for the adaptation and survival of C. sakazakii in the simulation of a newborn's stomach. This study gives directions for future research to further advance our understanding of the behavior of C. sakazakii in the GI tract of newborns., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cronobacter sakazakii is foodborne pathogen that causes serious illnesses such as necrotizing enterocolitis, meningitis and septicemia in infants. However, the virulence determinants and mechanisms of pathogenicity of these species remain unclear. In this study, multilocus sequence typing (MLST) was performed on 34 C. sakazakii strains and two strains with the same sequence type (ST) but distinct adhesion/invasion capabilities were selected for identification of differentially expressed proteins using data-independent acquisition (DIA) proteomic analysis. A total of 2,203 proteins were identified and quantified. Among these proteins, 210 exhibited differential expression patterns with abundance ratios ≥3 or ≤0.33 and P values ≤0.05. Among these 210 proteins, 67 were expressed higher, and 143 were expressed lower in C. sakazakii SAKA80220 (strongly adhesive/invasive strain) compared with C. sakazakii SAKA80221 (weakly adhesive/invasive strain). Based on a detailed analysis of the differentially expressed proteins, the highly expressed genes involved in flagellar assembly, lipopolysaccharide synthesis, LuxS/AI-2, energy metabolic pathways and iron-sulfur cluster may be associated with the adhesion/invasion capability of C. sakazakii. To verify the accuracy of the proteomic results, real-time qPCR was used to analyze the expression patterns of some genes at the transcriptional level, and consistent results were observed. This study, for the first time, used DIA proteomic to investigate potential adhesion/invasion related factors as a useful reference for further studies on the pathogenic mechanism of C. sakazakii.

Background and AimCronobacter sakazakii (C. sakazakii) has attracted considerable attention as an emerging neonatal pathogen and has been associated with outbreaks of life-threatening septicemia, necrotizing enterocolitis, and meningitis in neonates and infants globally. No data about the role of C. sakazakii as a cause of neonatal sepsis in North Africa is availale. Herein, we aimed to study the incidence of C. sakazakii in cases of neonatal sepsis, its distribution in different food samples in Egypt, antimicrobial profile, and the ability of the strains to form biofilms.MethodsA total of 100 positive blood cultures from cases of neonatal sepsis admitted to the neonatal ICU at Assiut University Children’s Hospital, Egypt, were analyzed. In addition, 1,100 food samples, including 400 powdered infant formula (PIF), 500 herbs, and 200 water samples were screened for the presence of C. sakazakii. We evaluated the antimicrobial profile and the ability of the strains to form biofilms.ResultsCronobacter sakazakii was detected in 12 out of 100 cases of neonatal sepsis. The organism was also isolated from PIF, herbs, and water in percentages of 17.5, 9.2, and 7.5%, respectively. Regarding the antimicrobial sensitivity, all strains were resistant to ampicillin, amoxicillin, ampicillin/sulbactam, clindamycin, cephalothin, and cephalexin. In addition, C. sakazakii strains showed the ability to form biofilms, but with variable degrees of cell density.ConclusionWe reported, for the first time, cases of neonatal sepsis caused by the emerging life-threatening pathogen C. sakazakii in Egypt. The organism was also detected in contaminated PIF, herbs, and water, which may be sources of infection for neonates, especially in countries where natural herbs are widely used as an alternative medicine. Finally, collective efforts by the parents, manufacturers of PIF, and healthcare personnel are essential to prevent serious infections caused by C. sakazakii, particularly in infants.

R ice based products could be have microbiological and toxicological hazards causing for unsuitable production and storage conditions. In this study, samples of three different commercial Infant Rice-based formulae with four samples IRF , nine milled rice MR samples were searched for Cronobacter sakazakii, Enterobacteriaceae spp. and Ochratoxin-A OTA . The presence of C. sakazakii and Enterobacteriaceae spp. was detected using ISO/TS 22964/IDF/RM 210 and and ISO 21528-2: 2004 method, respectively. A developed method using OCHRAPREP® REFN: A9-P14.V4 in conjunction with HPLC described by AOAC 2000.003 was used with slight modifications of the purification and HPLC conditions and with another confirmation method for OTA. The limit of quantities measurement was 1.286 μg/kg. C. sakazakii was not found in any samples. Enterobacteriaceae spp. was determined above the legal limits between 2.6x102 and 2.8x104 kob/g, average 3.46x104 in four samples 30.77% . OTA was detected by one IRF with milk product sample with 0.60 μg/kg 7.69%

Cronobacter sakazakii is foodborne pathogen that causes serious illnesses such as necrotizing enterocolitis, meningitis and septicemia in infants. However, the virulence determinants and mechanisms of pathogenicity of these species remain unclear. In this study, multilocus sequence typing (MLST) was performed on 34 C. sakazakii strains and two strains with the same sequence type (ST) but distinct adhesion/invasion capabilities were selected for identification of differentially expressed proteins using data-independent acquisition (DIA) proteomic analysis. A total of 2,203 proteins were identified and quantified. Among these proteins, 210 exhibited differential expression patterns with abundance ratios ≥3 or ≤0.33 and P values ≤0.05. Among these 210 proteins, 67 were expressed higher, and 143 were expressed lower in C. sakazakii SAKA80220 (strongly adhesive/invasive strain) compared with C. sakazakii SAKA80221 (weakly adhesive/invasive strain). Based on a detailed analysis of the differentially expressed proteins, the highly expressed genes involved in flagellar assembly, lipopolysaccharide synthesis, LuxS/AI-2, energy metabolic pathways and iron-sulfur cluster may be associated with the adhesion/invasion capability of C. sakazakii. To verify the accuracy of the proteomic results, real-time qPCR was used to analyze the expression patterns of some genes at the transcriptional level, and consistent results were observed. This study, for the first time, used DIA proteomic to investigate potential adhesion/invasion related factors as a useful reference for further studies on the pathogenic mechanism of C. sakazakii. [ABSTRACT FROM AUTHOR]

Background and Aim: Cronobacter sakazakii (C. sakazakii) has attracted considerable attention as an emerging neonatal pathogen and has been associated with outbreaks of life-threatening septicemia, necrotizing enterocolitis, and meningitis in neonates and infants globally. No data about the role of C. sakazakii as a cause of neonatal sepsis in North Africa is availale. Herein, we aimed to study the incidence of C. sakazakii in cases of neonatal sepsis, its distribution in different food samples in Egypt, antimicrobial profile, and the ability of the strains to form biofilms. Methods: A total of 100 positive blood cultures from cases of neonatal sepsis admitted to the neonatal ICU at Assiut University Children's Hospital, Egypt, were analyzed. In addition, 1,100 food samples, including 400 powdered infant formula (PIF), 500 herbs, and 200 water samples were screened for the presence of C. sakazakii. We evaluated the antimicrobial profile and the ability of the strains to form biofilms. Results: Cronobacter sakazakii was detected in 12 out of 100 cases of neonatal sepsis. The organism was also isolated from PIF, herbs, and water in percentages of 17.5, 9.2, and 7.5%, respectively. Regarding the antimicrobial sensitivity, all strains were resistant to ampicillin, amoxicillin, ampicillin/sulbactam, clindamycin, cephalothin, and cephalexin. In addition, C. sakazakii strains showed the ability to form biofilms, but with variable degrees of cell density. Conclusion: We reported, for the first time, cases of neonatal sepsis caused by the emerging life-threatening pathogen C. sakazakii in Egypt. The organism was also detected in contaminated PIF, herbs, and water, which may be sources of infection for neonates, especially in countries where natural herbs are widely used as an alternative medicine. Finally, collective efforts by the parents, manufacturers of PIF, and healthcare personnel are essential to prevent serious infections caused by C. sakazakii , particularly in infants. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii (C. sakazakii) can cause extremely high mortality diseases especially in infants, so it is necessary to rapidly and specifically detect C. sakazakii in powdered infant formula (PIF). In this work, the amplification strategy of methylene blue (MB)-anchored graphene oxide (GO) was used to develop an electrochemical aptasensor for detecting C. sakazakii easily and quickly. In the absence of C. sakazakii, GO would be bound to the aptamer of C. sakazakii by π-π interaction, and then adsorbed MB through electrostatic adherence; thus, the electrochemical signal of MB amplified. While in the presence of C. sakazakii, the bacteria would be specifically combined with the aptamer, and hindered the electrostatic adsorption of GO and MB to the aptamer, resulting in the decreasing electrochemical signal of MB. The fabricated electrochemical aptasensor showed a wide linear range from 2 × 101 to 2 × 106 CFU mL−1 of the bacteria concentration, and its limit of detection reached 7 CFU mL−1 (S/N = 3). In addition, the applicability of the aptasensor in PIF for the analysis of C. sakazakii was successfully demonstrated. This aptasensor would provide a new method for the detection of C. sakazakii but also a novel sensing platform for the rapid detection of pathogen bacteria. [ABSTRACT FROM AUTHOR]

The present study investigated inactivation of Cronobacter sakazakii inoculated on surface materials by bubble water–aided 405 nm light combined with folic acid (LFA). Polypropylene and silicon, widely used in baby feeding bottles, were used as surface materials, and inactivation of pathogens from surface materials and from water containing pathogens detached from surface materials was investigated. The combination of 405 nm light with folic acid accelerated the reduction of pathogens compared with all individual treatments. Even though the reduction of C. sakazakii inoculated on polypropylene was higher than that inoculated on silicon, significant differences were not observed for most experimental conditions. When folic acid was re-used for the maximum five times, the reduction of C. sakazakii inoculated on polypropylene and silicon did not differ significantly. Studies on the bactericidal mechanism of the combined treatment with reactive oxygen species scavengers revealed that hydrogen peroxide, singlet oxygen, and superoxide had significant effects on inactivation of C. sakazakii. When powdered infant formula (with and without lactic acid bacteria) was used as interference materials, reduction levels decreased compared to the control. The results of the present study showed that bubble water–aided combined treatment of 405 nm light and folic acidcan be used effectively to inactivate C. sakazakii on surface materials. • Bubble water aided LFA treatments showed synergistic bactericidal effect. • Reduction levels were not significantly different between PP and silicon. • Folic acid can be re-used 5 times maintaining the bactericidal efficacy. • ROS produced by LFA contributed significantly on the pathogen inactivation. • Powdered infant formula w or w/o LAB interfere the efficacy of LFA treatments. [ABSTRACT FROM AUTHOR]

Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter, however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii, C. malonaticus, and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.

Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter , however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii , C. malonaticus , and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites. [ABSTRACT FROM AUTHOR]

The effect of the cell-free culture supernatants (CFCSs) from different Lacobacillus spp. on growth ability of Cronobacter sakazakii ATCC 29544 was investigated by time-killing studies. The antimicrobial effect was evaluated using crude and 2.5 × concentrated CFCSs. Most of the CFCSs showed a dose-dependent antimicrobial activity, with the greatest C. sakazakii growth inhibition exerted by the CFCS 2.5 × of Lactobacillus casei rhamnosus ATCC 7469. Indeed, C. sakazakii growth was completely inhibited after 4 h of incubation with the crude CFCSs of L. casei rhamnosus and Lactobacillus acidophilus and after only 2 h using the related 2.5 × CFCSs. The flow cytometric analysis revealed that CFCSs altered the permeability of C. sakazakii cell membrane, showing 55% of live cells after 30 min of treatment with 2.5 × CFCSs of L. casei rhamnosus and L. acidophilus, reaching 1% of live cells after 2 h of exposure. The CFCSs of L. casei rhamnosus and L. acidophilus have showed anti-Cronobacter activity, determining a progressively inhibition of C. sakazakii growth as result of alterations in its membrane permeability. [ABSTRACT FROM AUTHOR]

ABSTRACT: Among the Cronobacter genus, Cronobacter sakazakii is the most common species posing a severe health risk for newborns, infants and children. Some infant formulas, cereal-based foods, and food production environments may be the potential reservoirs of C. sakazakii. This pathogen possesses different virulence factors encoded by different virulence genes. Therefore, characterizing these genes is important for distinguishing pathogenic strains from nonpathogenic ones. The objective of this study was to characterize some virulence genes [OmpA, OmpX, zpx, and Cpa] by real-time polymerase chain reaction (PCR) in C. sakazakii isolates from a total of 120 samples (20 each of milk powder, starch, rice flour, semolina, infant formula and dust samples from food production environments). Overall, 13 isolates (7 from milk powder, 2 rice flour, 1 semolina, and 3 dust) were cultured, identified by bioMérieux API® 20E test kit, and then subjected to real-time PCR application for screening the target virulence-associated genes. Our results showed that all of 13 isolates were positive for the virulence genes OmpA, OmpX, zpx, and Cpa. In summary, our study revealed that some of the analyzed foods and environmental samples were contaminated with pathogenic C. sakazakii with its virulence-associated markers, far above the allowable limit; and therefore, this level of contamination may pose a severe health threat for newborns, infants, and children.

Listeria monocytogenes and Cronobacter sakazakii are notorious pathogens involved in numerous foodborne outbreaks after ingested contaminated food. Bacteriocins are natural food preservatives, some of which have antimicrobial activity comparable with antibiotics. In this study, a plasmid encoded novel bacteriocin BMP11 produced by Lactobacillus crustorum MN047 was innovatively identified by combining complete genome and LC-MS/MS. The BMP11 was found to have rich α-helix conformation after prediction. Moreover, the antimicrobial activity of BMP11 was verified after its heterologous expression in E. coli with 1280 and 640 AU/mL against L. monocytogenes and C. sakazakii , respectively. After purification by anion-exchange chromatography and HPLC, BMP11 had MIC values of 0.3–38.4 μg/mL against tested foodborne pathogens. Further, it was found that BMP11 had bactericidal action mode with concomitant cell lysis to pathogens by growth curve and time-kill kinetics. The results of scanning electron microscope (SEM) and transmission electron microscope (TEM) indicated that BMP11 destroyed the integrity of cell envelope of pathogens with cell wall perforation and cell membrane permeabilization. The destruction of cell envelope integrity was further verified by propidium iodide (PI) uptake and lactic dehydrogenase (LDH) release. BMP11 increased inner-membrane permeability of C. sakazakii in a concentration-dependent manner. Meanwhile, BMP11 exhibited antibiofilm formation activity. In addition, BMP11 inhibited the growth of L. monocytogenes in milk. Therefore, BMP11 had promising potential as antimicrobial to control foodborne pathogens in dairy products. [ABSTRACT FROM AUTHOR]

Among the Cronobacter genus, Cronobacter sakazakii is the most common species posing a severe health risk for newborns, infants and children. Some infant formulas, cereal-based foods, and food production environments may be the potential reservoirs of C. sakazakii. This pathogen possesses different virulence factors encoded by different virulence genes. Therefore, characterizing these genes is important for distinguishing pathogenic strains from nonpathogenic ones. The objective of this study was to characterize some virulence genes [OmpA, OmpX, zpx, and Cpa] by real-time polymerase chain reaction (PCR) in C. sakazakii isolates from a total of 120 samples (20 each of milk powder, starch, rice flour, semolina, infant formula and dust samples from food production environments). Overall, 13 isolates (7 from milk powder, 2 rice flour, 1 semolina, and 3 dust) were cultured, identified by bioMérieux API® 20E test kit, and then subjected to real-time PCR application for screening the target virulence-associated genes. Our results showed that all of 13 isolates were positive for the virulence genes OmpA, OmpX, zpx, and Cpa. In summary, our study revealed that some of the analyzed foods and environmental samples were contaminated with pathogenic C. sakazakii with its virulence-associated markers, far above the allowable limit; and therefore, this level of contamination may pose a severe health threat for newborns, infants, and children. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii has been implicated in causing serious infections in neonates due to consumption of contaminated infant powdered milk. The zoonotic potential of the organism was not clear due to scarce evidence about the role of food animals in the transmission of infection. C. sakazakii was identified in infant powdered milk ( n = 100), infant stool ( n = 100), and dairy animal feces ( n = 100) with the percentages of 1%, 2%, and 4%, respectively. The outer membrane protein A ( ompA) gene was characterized in all isolates of different origin, while gene encoding for zinc-metaloprotease ( zpx) was only identified in isolates from animal feces. Genotyping of C. sakazakii isolates using enterobacterial repetitive intergenic consensus polymerase chain reaction revealed heterogenicity. The survival and thermotolerance of one potentially virulent C. sakazakii isolate of animal origin were examined at different temperatures. The isolate could survive with a stationary number at refrigeration temperature and the number increased significantly at room temperature after 24 h. The isolate showed thermoresistance when subjected to temperature range from 54°C to 64°C with D values ranged from 13.79 and 4.64 min and z value of 14.42. To the best of our knowledge, this is the first report of C. sakazakii isolation from buffalo feces in Egypt. [ABSTRACT FROM AUTHOR]

Emerging food-borne pathogens Cronobacter sakazakii, Klebsiella pneumoniae and Enterococcus faecalis may cause food-borne diseases in neonates, elderly and immunocompromised patients. Data on prevalence of emerging pathogens in food from India is lacking. Sixty food samples were analyzed from local markets in Mumbai for the presence of C. sakazakii, K. pneumoniae and E. faecalis. Present study shows that incidence of C. sakazakii and K. pneumoniae was highest in sprouts and least in milk (Table 1). Total 35% and 85% of food samples were positive for C. sakazakii and K. pneumoniae respectively. Culture independent species specific PCR based method was better than conventional culture based method in detecting these pathogens. E. faecalis was not detected in any of the food samples. All the isolates of C. sakazakii and K. pneumoniae were resistant to ampicillin. C. sakazakii was also resistant to cephalothin and K. pneumoniae was resistant to many other beta-lactam antibiotics like amikacin, aztreonam and amoxicillin-clavulanic acid. Our results suggest that conventional method should be carried out in combination with molecular methods for the accurate identification of these pathogens. Increase in drug resistance amongst emerging pathogens is also a concern. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii is an opportunistic pathogen that causes severe infections in neonates and infants through contaminated powdered infant formula (PIF). Therefore, the aim of this study was a large-scale study on determine the prevalence, molecular characterization and antibiotic susceptibility of C. sakazakii isolates from PIF purchased from Chinese retail markets. Two thousand and twenty PIF samples were collected from different institutions. Fifty-six C. sakazakii strains were isolated, and identified using fusA sequencing analysis, giving a contamination rate of 2.8%. Multilocus sequence typing (MLST) was more discriminatory than other genotyping methods. The C. sakazakii isolates were divided into 14 sequence types (STs) by MLST, compared with only seven clusters by ompA and rpoB sequence analysis, and four C. sakazakii serotypes by PCR-based O-antigen serotyping. C. sakazakii ST4 (19/56, 33.9%), ST1 (12/56, 21.4%), and ST64 (11/56, 16.1%) were the dominant sequence types isolated. C. sakazakii serotype O2 (34/56, 60.7%) was the primary serotype, along with ompA6 and rpoB1 as the main allele profiles, respectively. Antibiotic susceptibility testing indicated that all C. sakazakii isolates were susceptible to ampicillin-sulbactam, cefotaxime, ciprofloxacin, meropenem, tetracycline, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. The majority of C. sakazakii strains were susceptible to chloramphenicol and gentamicin (87.5 and 92.9%, respectively). In contrast, 55.4% C. sakazakii strains were resistant to cephalothin. In conclusion, this large-scale study revealed the prevalence and characteristics of C. sakazakii from PIF in Chinese retail markets, demonstrating a potential risk for neonates and infants, and provide a guided to effective control the contamination of C. sakazakii in production process.

Cronobacter sakazakii and Escherichia coli O157:H7 are well known food-borne pathogens that can cause severe disease. The identification of new alternatives to heating to control these pathogens in foods, while reducing the impact on organoleptic properties and nutritional value, is highly desirable. In this study, nisin and its bioengineered variants, nisin V and nisin S29A, are used alone, or in combination with plant essential oils (thymol, carvacrol and trans-cinnamaldehyde) or citric acid, with a view to controlling C. sakazakii and E. coli O157:H7 in laboratory-based assays and model food systems. The use of nisin variants (30 μM) with low concentrations of thymol (0.015%), carvacrol (0.03%) and trans-cinnamaldehyde (0.035%) resulted in extended lag phases of growth compared to those for corresponding nisin A-essential oil combinations. Furthermore, nisin variants (60 μM) used in combination with carvacrol (0.03%) significantly reduced viable counts of E. coli O157:H7 (3-log) and C. sakazakii (4-log) compared to nisin A-carvacrol treatment. Importantly, this increased effectiveness translated into food. More specifically, sub-inhibitory concentrations of nisin variants and carvacrol caused complete inactivation of E. coli O157:H7 in apple juice within 3 h at room temperature compared to that of the equivalent nisin A combination. Furthermore, combinations of commercial Nisaplin and the food additive citric acid reduced C. sakazakii numbers markedly in infant formula within the same 3 h period. These results highlight the potential benefits of combining nisin and variants thereof with carvacrol and/or citric acid for the inhibition of Gram negative food-borne pathogens. [ABSTRACT FROM AUTHOR]

This study evaluated the antimicrobial and desiccation resistance of Cronobacter sakazakii and Cronobacter malonaticus isolates from powdered infant formula and processing environments. The antimicrobial susceptibility tests showed that the 70 Cronobacter strains, representing 19 sequence types, were susceptible to the most of the antibiotics except for amoxicillin-clavulanate, ampicillin, and cefazolin. Furthermore, the growth of six C. sakazakii and two C. malonaticus strains from different sequence types (STs) in hyperosmotic media was measured. The growth of the two C. sakazakii strains (CE1 and CE13) from the neonatal pathovars ST4 and ST8, were significantly higher (p < 0.05) than that of other strains. C. malonaticus strain CM35 (ST201) was the slowest grower in all strains, and most could not grow in more than 8% NaCl solution. Also the survival of these strains under desiccation conditions was followed for 1 year. The viable count of Cronobacter spp. under desiccation conditions was reduced on average by 3.02 log cycles during 1 year, with CE13 (ST8) being the most desiccation resistant strain. These results will improve our understanding of the persistence of the two closely related species C. sakazakii and C. malonaticus which are of concern for neonatal and adult health. [ABSTRACT FROM AUTHOR]

In this study the antibacterial activity of lactoferrin against Cronobacter sakasakii (C. sakasakii) as a foodborne pathogen was investigated. The bovine lactoferrin (bLf) was isolated and purified from bovine colostrum by FPLC chromatography and visualized using (SDS-PAGE). Purification efficiency was 90%. C. sakasakii was isolated from a total of 100 samples of infant formula milk powder (IFMP) samples collected randomly from Qena city groceries and supermarkets using HiCrome Enterobacter sakazakiiagar followed by biochemical testing. C. sakasakii was detected in 21% of the total samples examined. The antibacterial effect of bLF on C. sakasakii isolates was tested via performing disk diffusion method using Mueller-Hinton agar. The results revealed that bLF at concentration of 10mg/ml showed the maximum inhibitory effect whereas the least inhibitory effect was recorded at concentration of 1mg/ml. The results indicated that bLF may be useful for inhibition of C. sakasakii in infant formula through supplementation or fortification. More attention should be paid during manufacture and handling of IFMP. [ABSTRACT FROM AUTHOR]

This study aimed to screen the in vitro antimicrobial activity of 10 plant essential oils (EOs) against 4 Cronobacter sakazakii strains, and use these oils or their combination to control C. sakazakii cocktail at low (3 log10 CFU/ml) and high (6 log10 CFU/ml) contamination levels in reconstituted infant milk formula (RIMF). Cinnamon and fir oils were the most inhibitory to C. sakazakii strains with inhibition zone of 32 to 40 mm at 20 µl/disc (the minimum inhibitory concentrations were 0.16 and 0.625 µl/ml, respectively). The addition of each of cinnamon or fir oil at 1% (v/v) reduced the C. sakazakii numbers in RIMF by 0.7-0.8 log10 CFU/ml when inoculated with high contamination level and by 2.5-3.1 log10 CFU/ml when inoculated with low contamination level. However, the combination of cinnamon and fir oils reduced C. sakazakii numbers at both inoculum levels to undetectable levels after 3 h of incubation at 37°C. The results of the current study indicated that a combination of cinnamon and fir oils has a potent antimicrobial activity which may potentially be used to control C. sakazakii in RIMF.

This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii ( C. sakazakii ), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti- C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti- C. sakazakii IgG to the magnetic nanoparticles was 86.23±0.59%. The assay developed in this study detected as few as 3.3×10 3 CFU mL −1 of C. sakazakii in pure culture within 2 h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2×10 5 CFU mL −1 of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10 3 CFU mL −1 of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii . [ABSTRACT FROM AUTHOR]

Copyright of Journal of Southern Agriculture is the property of Journal of Southern Agriculture and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cronobacter sakazakii and Salmonella occur at low levels in infant formula and survive desiccation and known to grow in reconstituted formula to infectious levels causing severe diseases in newborns. This research determined the minimum inhibitory (MIC) and bactericidal (MBC) concentrations of vanillin against nondesiccated and desiccated cocktails of C. sakazakii and Salmonella at 4, 10 and 23 °C in quarter-strength TSBYE. Next, the antimicrobial effect of several vanillin concentrations was tested against nondesiccated and desiccated cells at high and low inocula levels in reconstituted infant milk formula (IMF) at 4, 10 and 23 °C. The results showed that nondesiccated C. sakazakii and Salmonella were more susceptible to vanillin than desiccated cells at 4, 10, and 23 °C in ¼ TSBYE. At high inoculum level, vanillin inhibited the growth of nondesiccated C. sakazakii and Salmonella in reconstituted IMF at 4, 10 and 23 °C, but inhibited the growth of desiccated cells at 10 and 23 °C. At low inoculum level, vanillin inhibited the growth of nondesiccated and desiccated Salmonella at 4, 10, and 23 °C, but inhibited nondesiccated C. sakazakii at 4, 10, and 23 °C and inhibited desiccated cells at 23 °C. Generally, the inhibitory effect of vanillin against low inoculum was greater than that against high inoculum for nondesiccated and desiccated C. sakazakii and Salmonella . Thus, using vanillin as antimicrobial additive can control C. sakazakii and Salmonella in IMF. [ABSTRACT FROM AUTHOR]

Abstract: Cronobacter sakazakii is a foodborne pathogenic microorganism associated with sporadic cases of neonatal meningitis, necrotising enterocolitis, septicaemia, bloody diarrhoea and brain abscesses acquired through the consumption of contaminated powdered infant formula (PIF). This study aimed to investigate the growth of C. sakazakii DPC6529, a particularly stress tolerant clinical isolate, in acidified laboratory media and PIF. The possibility of a stationary-phase acid tolerance response (ATR) was also investigated. C. sakazakii DPC6529 grew in LB broth acidified to pH4.2 with hydrochloric acid (HCl) and was capable of relatively fast growth in PIF acidified to pH5.0 with HCl, representing the stomach pH reported for newborns and infants. Moreover, bacterial growth in LB broth supplemented with 1% (w/v) glucose gave rise to a stationary-phase ATR which resulted in enhanced survival against a subsequent acid challenge at pH3.0. A transposon mutagenesis approach was used to shed light on some of the molecular mechanisms involved in the response C. sakazakii DPC6529 to normally lethal acid exposures. The data suggests that repairing damage in proteins and nucleic acids, posttranscriptional modification of tRNA molecules and maintenance of the integrity of the cellular envelope are key processes in the defence against acid stress. Clones carrying transposon insertions in genes encoding the envelope stress response regulators CpxR and OmpR were identified as acid-sensitive mutants. Further analyses of the ompR defective mutant and its complemented counterpart evidenced that OmpR is a key player in the response of C. sakazakii to acid stress, although it was not essential to mount an active stationary-phase ATR, at least under the tested conditions. The ability of C. sakazakii DPC6529 to grow in acid environments and to develop an adaptive stationary-phase ATR may allow for its survival or even proliferation within the infant gastrointestinal tract after consumption of contaminated milk formulae. [Copyright &y& Elsevier]

Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p = 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii is an opportunistic pathogen that can cause bacteremia, meningitis, and necrotizing enterocolitis, most often in neonates with case-fatality rates that may reach 80%. The antimicrobial activity of lactic acid bacteria against a wide range of foodborne pathogens is well-established in different types of food products. The objective of the current study was to investigate the antibacterial activity of Lactobacillus acidophilus and L. casei isolated from feces of healthy infants against different strains of C. sakazakii in agar and a rehydrated infant milk formula (RIMF) model. The inhibition zones of C. sakazakii around L. acidophilus or L. casei ranged from 22 to 32 mm on eMan Rogosa Sharpe (MRS) agar under aerobic conditions, while a slight reduction in antibacterial activity was noted on modified MRS (0.2% glucose) under anaerobic conditions. It was observed that pH-neutralized cell-free supernatant (CFS) of L. acidophilus or L. casei was inhibitory against tested C. sakazakii strains. The inhibition zones of neutralized CFS were lower than the antibacterial activities of live cultures. The antibacterial activity of CFS was abolished when CFS from L. acidophilus or L. casei was heated at 60 or 80 °C for either 10 min or 2 h, or treated with trypsin or pepsin. This was considered strong evidence that the inhibition was due to the production of bacteriocins by L. casei and L. acidophilus. Both the CFS and active growing cells of L. casei and L. acidophilus were able to reduce the viability of C. sakazakii in the RIMF model. The results may extend the use of natural antimicrobials instead of conventional preservation methods to improve the safety of RIMF. [ABSTRACT FROM AUTHOR]